Requirements for cell surface expression of the influenza hemagglutinin (HA) were studied using a derived recombinant of SV40 which had full-length DNA sequences coding for the influenza virion HA inserted into the late region of SV40 DNA and propagated in the presence of tsA SV40 helper. Infection of primate cells with the HA-SV40 recombinant produced a functional glycosylated HA polypeptide that accumulated on the cell surface. To delineate the protein domains necessary for surface expression of the HA polypeptide, mutations were engineered in the recombinant HA-SV40 DNA. Deletion of the entire region coding for the hydrophobic COOH-terminus of the HA protein produced an HA polypeptide present first as a truncated glycosylated intracellular protein and later as a larger glycosylated secreted protein. The 2 proteins differed in their sensitivity to endoglycosidase H. A frameshift mutation at the beginning of the hydrophobic COOH-terminus introduced six charged amino acid residues. The resulting HA polypeptide was similar in size to and had the same endoglycosidase H sensitivity as unaltered HA. It was not secreted, was not on the cell surface, and was only present intracellularly. These findings suggest a role for the hydrophobic COOH-terminus not only in cell surface expression but the secretion related glycosylation.